The present invention relates to novel recombinant vaccines providing protective immunity especially against tuberculosis. Further, the present invention refers to novel recombinant nucleic acid molecules, vectors containing said nucleic acid molecules, cells transformed with said nucleic acid molecules and polypeptides encoded by said nucleic acid molecules.
Tuberculosis (TB) caused by Mycobacterium tuberculosis remains a significant global problem. It is estimated that one third of the world""s population is infected with M.tuberculosis (Kochi, 1991). In many countries the only measure for TB control has been vaccination with M.bovis bacille Calmette-Guxc3xa9rin (BCG). The overall vaccine efficacy of BCG against TB, however, is about 50% with extreme variations ranging from 0% to 80% between different field trials (Roche et al., 1995). Thus, BCG should be improved, e.g. by genetic engineering, to provide a vaccine for better TB control (Murray et al., 1996; Hess and Kaufmann, 1993). The widespread emergence of multiple drug-resistant M.tuberculosis strains additionally underlines the urgent requirement for novel TB vaccines (Grange, 1996).
M.tuberculosis belongs to the group of intracellular bacteria that replicate within the phagosomal vacuoles of resting macrophages, thus protection against TB depends on T cell-mediated immunity (Kaufmann, 1993). Several studies in mice and humans, however, have shown that mycobacteria stimulate antigen-specific, major histocompatibility complex (MHC) class II- or class I-restricted CD4 and CD8 T cells, respectively (Kaufmann, 1993).
The important role of MHC class I-restricted CD8 T cells was convincingly demonstrated by the failure of xcex22-microglobulin (xcex22m) deficient mice to control experimental M.tuberculosis infection (Flynn et al., 1993). Because these mutant mice lack MHC class I, functional CD8 T cells cannot develop. In contrast to M.tuberculosis infection, xcex22m-deficient mice are capable of controlling certain infectious doses of the BCG vaccine strain (Flynn et al., 1993; Ladel et al., 1995). Furthermore, BCG vaccination of xcex22m-deficient mice prolonged survival after subsequent M.tuberculosis infection whereas BCG-immunized C57BL/6 resisted TB (Flynn et al., 1993). This differential CD8 T cell dependency between M.tuberculosis and BCG may be explained as follows: M.tuberculosis antigens gain better access to the cytoplasm than antigens from BCG leading to more pronounced MHC class I presentation (Hess and Kaufmann, 1993). Consequently, a more effective CD8 T cell response is generated by M.tuberculosis. This notion was recently supported by increased MHC class I presentation of an irrelevant antigen, ovalbumin, by simultaneous M.tuberculosis, rather than BCG, infection of antigen presenting cells (APC) (Mazzaccaro et al., 1996).
Secreted proteins of M.tuberculosis comprise a valuable source of antigens for MHC class I presentation. Recently, a DNA vaccine encoding the secreted antigen Ag85A elicited MHC class I-restricted CD8 T cell responses in mice which may contribute to defence against TB (Huygen et al., 1996). In general, evidence is accumulating that immunization with secreted protein antigens of M.tuberculosis induce some protection against TB in guinea pigs and mice (Horwitz et al., 1995; Andersen, 1994). An important goal towards the development of improved TB vaccines based on BCG, therefore, is to augment the accessibility of secreted BCG-specific antigens to the cytoplasm of infected APC. Subsequent delivery of peptides derived from these secreted proteins into the MHC class I presentation pathway may potentiate the already existing BCG-specific immune response for preventing TB.
The phagolysosomal escape of L.monocytogenes represents a unique mechanism to facilitate MHC class I antigen presentation of listerial antigens (Berche et al., 1987; Portnoy et al., 1988). Listeriolysin (Hly), a pore-forming sulfhydryl-activated cytolysin, is essential for the release of L.monocytogenes microorganisms from phagolysosomal vacuoles into the cytosol of host cells (Gaillard et al., 1987; Portnoy et al., 1988). This escape function was recently transferred to Bacillus subtilis and to attenuated Salmonella ssp. strains (Bielecki et al., 1991; Gentschev et al., 1995; Hess and Kaufmann, 1997). Hly expression by an asporogenic B.subtilis mutant strain or in Salmonella ssp. results in bacterial escape from the phagolysosome into the cytosol of J774 macrophage-like cells (Bielecki et al., 1991; Gentschev et al., 1995; Hess and Kaufmann, 1997).
Thus, the transfer of lysosomal escape functions to heterologous microorganisms may cause an elevated toxicity of the resulting recombinant microorganisms. For this reason, the use of these lysosomal escape functions for the preparation of recombinant living vaccines has not been readily taken into consideration.
According to the present invention recombinant BCG strains secreting hemolytically active Hly were constructed which show an improved efficacy MHC class I-restricted immune response and, surprisingly, an equal or even lower cytotoxicity in comparison with the unmodified native BCG strains. Thus, these recombinant organisms are promising candidate vaccines against TB.
A first aspect of the present invention is a recombinant nucleic acid molecule encoding a fusion polypeptide comprising (a) at least one domain from a Mycobacterium polypeptide, wherein said domain is capable of eliciting an immune response in a mammal, and (b) a phagolysosomal escape domain.
A specific embodiment of this first aspect is the nucleic acid molecule in SEQ ID No.1. This nucleic acid molecule comprises a signal peptide coding sequence (nucleotide 1-120), a sequence coding for an immunogenic domain (nucleotide 121-153), a peptide linker coding sequence (nucleotide 154-210), a sequence coding for a phagolysosomal domain (nucleotide 211-1722), a further peptide linker coding sequence (nucleotide 1723-1800) and a sequence coding for a random peptide (nucleotide 1801-1870). The corresponding amino acid sequence is shown in SEQ ID No.2.
The nucleic acid of the present invention contains at least one immunogenic domain from a polypeptide derived from an organism of the genus Mycobacterium, preferably from Mycobacterium tuberculosis or from Mycobacterium bovis. This domain has a length of at least 6, preferably of at least 8 amino acids. The immunogenic domain is preferably a portion of a native Mycobacterium polypeptide. However, within the scope of the present invention is also a modified immunogenic domain, which is derived from a native immunogenic domain by substituting, deleting and/or adding one or several amino acids.
The immunogenic domain is capable of eliciting an immune response in a mammal. This immune response can be a B cell-mediated immune response. Preferably, however, the immunogenic domain is capable of eliciting a T cell-mediated immune response, more preferably a MHC class I-restricted CD8 T cell response.
The domain capable of eliciting an immune response is peferably selected from immunogenic peptides or polypeptides from M.bovis or M.tuberculosis or from immunogenic fragments thereof. Specific examples for suitable antigens are Ag85B (p30) from M.tuberculosis (Harth et al., 1996), Ag85B (xcex1-antigen) from M.bovis BCG (Matsuo et al., 1988), Ag85A from M.tuberculosis (Huygen et al., 1996) and ESAT-6 from M.tuberculosis (Sorensen et al., 1996, Harboe et al., 1996 and Andersen et al., 1995). More preferably, the immunogenic domain is derived from the antigen Ag85B. Most preferably, the immunogenic domain comprises the sequence from aa.41 to aa.51 in SEQ ID No.2.
The recombinant nucleic acid molecule according to the present invention further comprises a phagolysosomal escape domain, i.e. a polypeptide domain which provides for an escape of the fusion polypeptide from the phagolysosome into the cytosol of mammalian cells. Preferably, the phagolysosomal escape domain is derived from an organism of the genus Listeria. More preferably, the phagolysosomal escape domain is derived from the organism L.monocytogenes. Most preferably, the phagolysosomal domain is encoded by a nucleic acid molecule selected from: (a) the nucleotide sequence from nucleotide 211-1722 as shown in SEQ ID No.1, (b) a nucleotide sequence which encodes for the same amino acid sequence as the sequence from (a), and (c) a nucleotide sequence hybridizing under stringent conditions with the sequence from (a) or (b).
Apart from the nucleotide sequence depicted in SEQ ID No.1 the present invention also comprises nucleic acid sequences hybridizing therewith. In the present invention the term xe2x80x9chybridizationxe2x80x9d is used as defined in Sambrook et al. (Molecular Cloning. A laboratory manual, Cold Spring Harbor Laboratory Press (1989), 1.101-1.104). In accordance with the present invention the term xe2x80x9chybridizationxe2x80x9d is used if a positive hybridization signal can still be observed after washing for one hour with 1xc3x97SSC and 0.1% SDS at 55xc2x0 C., preferably at 62xc2x0 C. and more preferably at 68xc2x0 C., particularly for 1 hour in 0.2xc3x97SSC and 0.1% SDS at 55xc2x0 C., preferably at 62xc2x0 C. and more preferably at 68xc2x0 C. A sequence hybridizing with a nucleotide sequence as per SEQ ID No.1 under such washing conditions is a phagolysosomal escape domain encoding nucleotide sequence preferred by the subject invention.
Preferably, the recombinant nucleic acid molecule encoding for a fusion polypeptide contains a signal peptide encoding sequence. More preferably, the signal sequence is a signal sequence active in Mycobacteria, preferably in M.bovis, e.g. a native M.bovis signal sequence. A preferred example of a suitable signal sequence is the nucleotide sequence coding for the Ag85B signal peptide which is depicted in SEQ ID No.1 from nucleotide 1 to 120.
Further, it is preferred that a peptide linker be provided between the immunogenic domain and the phagolysosomal escape domain. Preferably, said peptide linker has a length of from 5 to 50 amino acids. More preferably, a sequence encoding a linker as shown in SEQ ID No.1 from nucleotide 154 to 210 or a sequence corresponding thereto as regards the degeneration of the genetic code.
A further subject matter of the invention pertains to a recombinant vector comprising at least one copy of a nucleic acid molecule as defined above. Preferably, the recombinant vector is a prokaryotic vector, i.e. a vector containing elements for replication or/and genomic integration in prokaryotic cells. Preferably, the recombinant vector carries the nucleic acid molecule of the present invention operatively linked with an expression control sequence. The expression control sequence is preferably an expression control sequence active in Mycobacteria, particularly in M.bovis. The vector can be an extrachromosomal vector or a vector suitable for integration into the chromosome. Examples of such vectors are known to the man skilled in the art and, for instance, given in Sambrook et al. supra.
A still further subject matter of the invention is a cell comprising a recombinant nucleic acid molecule or a vector as defined above. Preferably, the cell is prokaryotic, particularly a Mycobacterium cell. Further, it is preferred that the cell is capable of expressing the nucleic acid molecule of the invention.
In a second aspect of the present invention a recombinant Mycobacterium bovis cell is provided which comprises at least one recombinant nucleic acid molecule encoding a fusion polypeptide comprising (a) at least one domain capable of eliciting an immune response in a mammal and (b) a phagolysosomal escape domain. According to this aspect, the immunogenic domain is not restricted to Mycobacterium antigens and can be selected from autoantigens, tumor antigens and pathogen antigens such as virus antigens, parasite antigens, bacterial antigens in general and immunogenic fragments thereof. Specific examples for suitable tumor antigens are human tumor antigens such as the p53 tumor suppressor gene product (Houbiers et al., 1993) and melanocyte differentiation antigens, e.g. Melan-A/MART-1 and gp100 (van Elsas et al., 1996). Specific examples for suitable virus antigens are human tumor virus antigens such as human papilloma virus antigens, e.g. antigens E6 and E7 (Bosch et al., 1991), influenza virus antigens, e.g. influenza virus nucleoprotein (Matsui et al., 1995; Fu et al., 1997) or retroviral antigens such as HIV antigens, e.g. the HIV-1 antigens p17, p24, RT and Env (Harrer et al., 1996; Haas et al., 1996). Specific examples for suitable parasite antigens are Plasmodium antigens such as liver stage antigen (LSA-1), circumsporozoite protein (CS or allelic variants cp26 or cp29), thrombospondin related amonymous protein (TRAP), sporozoite threonine and asparagine rich protein (STARP) from Plasmodium falciparum (Aidoo et al., 1995) and Toxoplasma antigens such as p30 from Toxoplasma gondii (Khan et al., 1991; Bulow and Boothroyd, 1991). Specific examples for suitable bacterial antigens are Legionella antigens such as Major secretary protein from Legionella pneumophila (Blander and Horwitz, 1991).
The cell according to the invention is preferably capable of secreting the fusion polypeptide encoded by the nucleic acid molecule of the invention and of providing it in a form suitable for MHC class I-restricted antigen recognition.
In a third aspect of the present invention a recombinant Mycobacterium bovis cell is provided which comprises at least one nucleic acid molecule encoding a phagolysosomal escape peptide or polypeptide. Even if the phagolysosomal escape peptide or polypeptide is not fusioned with an antigen, a surprising improvement of the immunogenic properties is found.
The recombinant Mycobacterium bovis cell which is provided according to the present invention may contain at least one further recombinant, e.g. heterologous nucleic acid molecule encoding a peptide or polypeptide capable of eliciting an immune response in a mammal. Said further immunogenic peptide or polypeptide may be selected from Mycobacterium antigens or, in a wider sense, from autoantigens, tumor antigens, pathogen antigens and immunogenic fragments thereof. The nucleic acid molecule coding for the further peptide or polypeptide may be situated on the same vector as the fusion gene. However, it may, for example, also be situated on a different plasmid, independently of the fusion gene, or be chromosomally integrated.
Surprisingly, it was found that a Mycobacterium cell according to the present invention has an intracellular persistence in infected cells, e.g. macrophages, which is equal or less than the intracellular persistence of a corresponding native Mycobacterium cell which does not contain the recombinant nucleic acid molecule.
A still further subject matter of the present invention is a recombinant fusion polypeptide encoded by a nucleic acid molecule as defined above. The fusion polypeptide according to the invention imparts to a cell the capability of improved MHC class I-restricted antigen recognition.
The present invention also refers to a pharmaceutical composition comprising as an active agent a cell or a fusion polypeptide as defined above, optionally together with pharmaceutically acceptable diluents, carriers and adjuvants. Preferably, the composition is a living vaccine suitable for administration to a mammal, preferably a human. The actually chosen vaccination route depends on the choice of the vaccination vector. Administration may be achieved in a single dose or repeated at intervals. The appropriate dosage depends on various parameters such as the vaccinal vector itself or the route of administration. Administration to a mucosal surface (e.g. ocular, intranasal, oral, gastric, intestinal, rectal, vaginal or urinary tract) or via the parenteral route (e.g. subcutaneous, intradermal, intramuscular, intravenous or intraperitoneal) might be chosen.
Further, the present invention pertains to a method for preparing a recombinant bacterial cell as defined above. According to the first aspect, this method comprises the steps of (i) inserting a recombinant nucleic acid molecule into a bacterial cell, said nucleic acid molecule encoding a fusion polypeptide comprising (a) at least one domain from a Mycobacterium polypeptide wherein said domain is capable of eliciting an immune response in a mammal and (b) a phagolysosomal escape domain, and (ii) cultivating the cell obtained according to step (i) under suitable conditions. Preferably, a cell is obtained which is capable of expressing said nucleic acid molecule. Preferably, the cell is a M.bovis cell.
According to the second aspect, this method comprises the steps of (i) inserting a recombinant nucleic acid molecule into a Mycobacterium bovis cell, said nucleic acid molecule encoding a fusion polypeptide comprising (a) at least one domain from a polypeptide, wherein said domain is capable of eliciting an immune response in a mammal, and (b) a phagolysosomal escape domain, and (ii) cultivating the cell obtained according to (i) under suitable conditions.
According to the third aspect, this method comprises the step of (i) inserting a recombinant nucleic acid molecule into a Mycobacterium bovis cell, said nucleic acid molecule encoding a phagolysosomal escape peptide or polypeptide, and (ii) cultivating the cell obtained according to (i) under suitable conditions.
If desired, the method of the present invention comprises inserting at least one further recombinant nucleic acid molecule into the Mycobacterium bovis cell, said further recombinant nucleic acid molecule encoding a peptide or polypeptide capable of eliciting an immune response in a mammal.
Finally, the present invention relates to a method for the preparation of a living vaccine comprising formulating the recombinant cell in a pharmaceutically effective amount with pharmaceutically acceptable diluents, carriers and/or adjuvants.